Measurement of particle concentration is important in many industrial and research applications. For example, monitoring cell density (e.g. biomass) in liquid cell cultures is used: to determine the growth phase or rate; as a feedback signal for adjusting growth conditions (e.g. dissolved oxygen, pH, media constituents); and/or as an indicator of when to induce expression of genes, harvest cells, or inoculate cells into a larger media volume. The growth rate of many cultures, particularly microbial organisms (e.g. yeast, bacteria), is limited by the concentration of dissolved oxygen in the medium. The culture of such organisms is often performed in vessels (e.g. fermenters, bioreactors) in which gases are bubbled (“sparged”) and the medium is stirred or otherwise agitated, often at such high rates that the bubbles constitute a significant fraction (“gas hold-up”) of the total volume. The presence of such a high concentration of bubbles presents a challenge to many techniques for cell growth monitoring.
Many biomass monitoring techniques take advantage of the scattering of light by cells. For example, one of the most common laboratory techniques for monitoring cell growth is to extract a sample, dilute it, and measure it's absorbance (e.g. at 600 nm) in a fixed path length (e.g. 1 cm) cell in a spectrophotometer. Absorbance is typically limited to about 0.5 in order to remain in the linear range of Beer's Law. The measured absorbance multiplied by the dilution factor is referred to as the optical density (e.g. “OD(600 nm)”), and used as an indication of biomass. Despite its prevalence, this technique has numerous limitations: it requires opening the culture, with the attendant risk of contamination; the dilution step is subject to volumetric error; the extracted sample is expended, of particular concern in small volume cultures; and it is labor-intensive.
In an effort to overcome these limitations and provide continuous (“on-line”) monitoring, much work has gone into the development of invasive sensors for measuring optical density directly in the cell culture. Unfortunately, such sensors are subject to the same limitation of narrow linear range as are off-line techniques: in order to measure biomass over a wide range, the use of multiple sensors, having different optical transmission path lengths, is frequently required. Immersible sensors that measure back-reflected light (instead of transmission) typically have a somewhat wider but still limited linear range of response to biomass, and may suffer, particularly in the low biomass range, from a sensitivity to reflections from nearby non-biological objects within the vessel, such as impellers, pH sensors, etc. This can render the sensors inaccurate, in an unpredictable way.
Methods for monitoring biomass non-invasively, through the vessel wall, or through an optical window, have been developed in recent years. In U.S. Pat. No. 7,100,462 “Self Adjusting Sensor Mounting Device”, methods and devices are described for reproducibly mounting a sensor to a wide variety of cylindrical and flat surfaces in a manner that automatically compensates for the curvature of the mounting surface.
In U.S. Pat. No. 8,603,772, “Particle sensor with wide linear range”, methods and devices are described for measuring particulate concentration in vessels, where the response from multiple source-detector pairs is combined to provide a linear response over a wide range of particle concentrations. Also described, are methods and devices for confining the measurement to a specific volume within the medium, as methods and devices for performing rapid sequential measurement of particle concentration in multiple vessels.
In U.S. Pat. No. 8,405,033 “Optical sensor for rapid determination of particulate concentration”, methods and devices are described for limiting the optical penetration depth of measurements of particle density by the use of light at wavelengths that are strongly absorbed by the medium, and matching the source-detector separation to the absorbance path length.
In view of the above, a need exists for devices that can read particulate concentrations accurately in the presence of bubbles as well as other nearby reflective objects, that is not prone to fouling, and that is linear over a wide range of biomass. Benefits could also be realized from methods and devices capable of reading particle concentrations in shallow samples and without the need to dilute the sample, or employ multiple sensors. The present invention provides these and other features that will be apparent upon review of the following.